Generally speaking, this invention relates to a liquid formulation for maintaining urine specimens. More particularly, this invention relates to such a formulation wherein urine specimens are maintained from the addition of the specimen until such time as testing of the specimen takes place, without any other preservation methods such as refrigeration, and which formulation simultaneously prevents additional growth of bacteria present in the specimen so that a precise accurate specimen is presented for examination. This invention is related to the inventions described and claimed in U.S. patent application Ser. No. 3,237 filed Jan. 15, 1979, now U.S. Pat. No. 4,258,032, issued Mar. 24, 1981, and U.S. application Ser. No. 209,816 filed Nov. 24, 1980 now U.S. Pat. No. 4,336,880, issued June 29, 1982, both of which applications are hereby incorporated by reference in their entirety.
Bacterial quantitation of clean-voided urine specimens is employed to determine the presence of urinary tract infection. In many cases, however, such specimens are contaminated from exogenous sources. Also, in view of the fact that urine has the capability of supporting the proliferation of bacteria, multiplication of such contaminants may occur which will result in false positives. As a result, in order to prevent multiplication of contaminants, it has been recommended in the past that culturing take place of a urine specimen within two to four hours of the taking of the specimen, or that refrigeration be applied to the specimen. As will be appreciated, in many cases such culturing or refrigeration is not possible. Therefore, there is a need for a maintenance formula for urine specimens which is capable of maintaining and preserving the urine intact once the sample is taken, and until such time as the sample may be examined, and which maintenance formula prevents multiplication of bacterial contaminants. In the past, powdered boric acid has been proposed as a preservative. However, boric acid is toxic to test strains present in urine. Therefore, the addition of boric acid will provide inaccurate results in some cases. Moreover, such powdered boric acid is not effective for preventing proliferation of some bacterial contaminant strains.
By contrast, in accordance with the present invention, there is provided a liquid maintenance formula for urine specimens comprising distilled water, sorbitol, boric acid, or salts thereof, and an alkali formate. The components of the formula are maintained in the formula in an amount effective to maintain a urine specimen in its initial form. It has been found with the formula of the invention that a urine sample is maintained without proliferation of bacterial contaminants, and simultaneously, is not unduly toxic to the bacteria present in the specimen.
A modified formulation, also found to be effective in manufacturing urine specimens for transporting them over a period of time without refrigeration, includes mannitol, sodium borate, and boric acid in distilled water. This particular formulation has proved to be particularly effective for status quo maintenance of microbial systems containing streptococcus faecalis. Also, it will preserve urine specimens containing pseudomonas aeruginosa at ambient temperatures for up to 72 hours.
While the urine maintenance formulation described and claimed in the above noted applications has proved effective for maintaining urine specimens without refrigeration, the formulation has certain disadvantages in that the formulation cannot be lyophilized for certain applications. Moreover, the viscosity thereof may provide difficulties in handling in certain applications. Also, those formulations containing glycerin are light sensitive over a period of time. In addition, the formulation may not be sterilized by irradiation.
By contrast, the sorbitol formulation herein is a solution with a reduced viscosity providing a liquid which is more easily dispensed, and providing a more accurate volume control during dispensing. Moreover, it allows freeze drying of the solution, thus eliminating dilution of the urine, and providing a product which may be sterilized by irradiation.
Indeed, the modified formulation containing mannitol discussed above may be lyophilized rapidly as compared to the formulation containing sorbitol. Also, the stability of the formulations are increased and their light sensitive properties are reduced substantially. A further important advantage of the formulations are that the specific gravity of the urine remains within diagnostic tolerances when the sample is added to the maintenance fluid. For example, the formulations consistently raise the specific gravity only 0.006, whereas a glycerin containing formulation raises the specific gravity beyond readable limits in certain applications with a urine refractometer.
In considering generally the conditions for achieving the most enhanced results in connection herewith, which conditions are more specifically set forth below, one may note that satisfactory results have been achieved in accordance herewith with a sorbitol formulation containing distilled water within the range of between about 85 and 105 ml. and preferably 100 ml., sorbitol within the range of between about 15 and 25 gms., and prefereably 20 gms., boric acid or salts thereof, such as sodium borate within the range of between about 8 and 12 gms., and preferably 10 gms., and an alkali formate within the range of between about 4 and 6 gms. and preferably 5 gms. The alkali formate may be, for example, sodium formate.
The formulations may include as an additional component, a buffer. A representative buffer is sodium acetate for example, which may be present within the formulation within the range of between about 4 and 6 gms. and preferably 5 gms. Other buffers may be used such as organic buffers including tris hydroxymethyl amino methane. Also, inorganic phosphate buffers may be used.
Glutamine or other amino acids such as arginine may be included specifically for the purpose of facilitating maintenance of the Pseudomonas species. The glutamine or other amino acid may be present within the range of between about 0.15 and 0.25 gms. and preferably 0.20 gms.
Also, a surfactant such as a non-ionic polysorbate 80 USP, present in the amount within the range of between about 0.5 and 0.7 ml, and preferably 0.6 ml may be included in the formulations. Such a representative non-ionic surfactant may be, for example, a one percent solution of Tween 80, a polyoxyethylene sorbitan monoleate, a product of Atlas Corporation. Other surfactants may be, for example, amphoteric compounds containing carboxylate or phosphate groups such as, for example, proteins or polypeptides, lecithins or cephalins, or other non-ionic compounds such as fatty acid esters, propylene glycol, sorbitan, or sucrose.
With the mannitol containing formulation, satisfactory results are achieved with a formulation containing distilled water within the range of between about 85 and 115 ml., and preferably 100 ml., mannitol within the range of between about 9 and 11 grams, and preferably 10 grams, boric acid within the range of between about 4.5 and 5.5 grams, and preferably 5 grams, and sodium borate within the range of between about 10.8 and 13.2 grams, and preferably 12 grams.
One of the aspects of this invention to be considered in the context of the maintenance of a urine sample is that it is desirable, in accordance with this invention, to have a final percentage of the active components of the sorbitol maintenance formulation herein present in specific amounts in the sample once the addition of the urine sample is made. For example, preferably, sorbitol is present within the range of between about 1.5 and 2.5 percent in the combined urine and maintenance formula, boric acid is present within the range of between about 0.8 and 1.2 percent, and sodium formate is present within the range of between about 0.4 and 0.6 percent. If glutamine is used additionally, it will be present within the range of between about 0.015 and 0.025 percent. By the same token, if a surfactant is used such as for example, Tween 80, which is a one percent solution, the final percentage quantity present in the combined urine maintenance formula sample will be within the range of 0.0005 and 0.0007 percent. If a buffer is used such as sodium acetate, for example, it will be present within the range of between about 0.4 and 0.6 percent.
If, on the other hand, the mannitol containing formulation is used the various components should be present in a urine sample, as follows: assuming a 5 ml. sample of urine, then mannitol should be present in the same within the range of between about 0.9 and 1.1 percent of the urine sample; sodium borate should be present in the amount within the range of between about 1.08 and 1.32 percent, and boric acid should be present in the amount within the range of between about 0.45 and 0.55 percent.
As will be appreciated, a lesser quantity of components is required for the mannitol urine specimen maintenance formula in accordance with this invention, thus making it simpler and less expensive for formulate.
As purely illustrative of the results achieved by the maintenance formulas, in accordance herewith, examples were prepared using the preferred ratio of components in the sorbitol containing maintenance formula. It is to be understood, that these examples are being presented with the understanding that they are to have no limiting character on the broad disclosure of the invention as generally set forth herein and as directed to men skilled in the art.